A genetically modified minipig model for Alzheimer's disease with SORL1 haploinsufficiency.

The established causal genes in Alzheimer's disease (AD), APP, PSEN1, and PSEN2, are functionally characterized using biomarkers, capturing an in vivo profile reflecting the disease's initial preclinical phase. Mutations in SORL1, encoding the endosome recycling receptor SORLA, are found in 2%-3% of individuals with early-onset AD, and SORL1 haploinsufficiency appears to be causal for AD. To test whether SORL1 can function as an AD causal gene, we use CRISPR-Cas9-based gene editing to develop a model of SORL1 haploinsufficiency in Göttingen minipigs, taking advantage of porcine models for biomarker investigations. SORL1 haploinsufficiency in young adult minipigs is found to phenocopy the preclinical in vivo profile of AD observed with APP, PSEN1, and PSEN2, resulting in elevated levels of ß-amyloid (Aß) and tau preceding amyloid plaque formation and neurodegeneration, as observed in humans. Our study provides functional support for the theory that SORL1 haploinsufficiency leads to endosome cytopathology with biofluid hallmarks of autosomal dominant AD.

Reprint of: Nonclinical species sensitivity to convulsions: An IQ DruSafe consortium working group initiative.

Clinical development of compounds that carry a convulsion liability is typically limited by safety margins based on the most sensitive nonclinical species. To better understand differences in sensitivity to drug-induced convulsion of commonly used preclinical species, a survey was distributed amongst pharmaceutical companies through an IQ consortium (International Consortium for Innovation and Quality in Pharmaceutical Development) resulting in convulsion-related data on 80 unique compounds from 11 companies. The lowest free drug plasma concentration at which convulsions were observed and the no observed effect level for convulsions were compared between species to determine their relative sensitivity. Additionally, data were collected on other endpoints including use of electroencephalography, premonitory signs, convulsion type, the reason why development was stopped, and the highest development phase reached. The key outcomes were: (1) the dog was most often determined to be the most sensitive species by both non-exposure and exposure-based analyses, (2) there was not a clear sensitivity ranking of other species (NHP, rat and mouse), (3) CNS symptoms were frequently present at exposures that were not associated with convulsions, but no single reliable premonitory indicator of convulsion was identified, and (4) the lack of convulsions in the compounds that were tested in humans in this dataset may suggest that convulsion liability is well mitigated via current drug development strategies.

Nonclinical species sensitivity to convulsions: An IQ DruSafe consortium working group initiative.

Clinical development of compounds that carry a convulsion liability is typically limited by safety margins based on the most sensitive nonclinical species. To better understand differences in sensitivity to drug-induced convulsion of commonly used nonclinical species, a survey was distributed amongst pharmaceutical companies through an IQ consortium (International Consortium for Innovation and Quality in Pharmaceutical Development) resulting in convulsion-related data on 80 unique compounds from 11 companies. The lowest free drug plasma concentration at which convulsions were observed and the no observed effect level for convulsions were compared between species to determine their relative sensitivity. Additionally, data were collected on other endpoints including use of electroencephalography, premonitory signs, convulsion type, the reason why development was stopped, and the highest development phase reached. The key outcomes were: (1) the dog was most often determined to be the most sensitive species by both non-exposure and exposure-based analyses, (2) there was not a clear sensitivity ranking of other species (NHP, rat and mouse), (3) CNS symptoms were frequently present at exposures that were not associated with convulsions, but no single reliable premonitory indicator of convulsion was identified, and (4) the lack of convulsions when compounds were tested in humans in this dataset may suggest that convulsion liability is well mitigated via current drug development strategies.

Refinement of the rodent pentylenetetrazole proconvulsion assay, which is a good predictor of convulsions in repeat-dose toxicology studies.

INTRODUCTION: The pentylenetetrazole (PTZ)-induced seizure assay in rodents is an established method for investigating drug-induced alterations in seizure threshold such as proconvulsant effects. The standard procedure in our laboratory was to administer the test item prior to 75-120 mg/kg subcutaneous PTZ. However, this dose range is associated with a high incidence of mortality, including approximately 40% or greater deaths of control animals. METHODS: The predictivity of the PTZ-induced seizure assay was retrospectively evaluated by relating drug plasma levels associated with proconvulsant effects to exposures observed during convulsions in repeat-dose toxicology studies. Margins to estimated efficacious doses were also considered. To investigate potential refinements, a high PTZ dose (80 mg/kg, subcutaneously) was compared to two lower doses (40 and 60 mg/kg), and a range of doses of theophylline was orally administered as positive control. RESULTS: The PTZ-induced proconvulsion assay proved to be a good predictor of convulsions in toxicology studies. In the refinement study, theophylline potentiated PTZ-induced seizures over all doses tested. At 60 mg/kg PTZ, the proconvulsant dose-dependency of theophylline was best observed. At both 40 and 60 mg/kg PTZ, mortality in control animals was significantly reduced. DISCUSSION: Risk assessment at an early stage of drug development supports candidate selection and, along that approach, the PTZ proconvulsion assay was proven to be a good predictor of convulsions in subsequent toxicology studies. It was also demonstrated that a relatively lower PTZ dose (60 mg/kg) improved the dose-response-curve of the positive control tested, decreased mortality overall and, therefore, contributes to refining this standard procedure for CNS safety evaluation.

Exploratory EEG studies for the assessment of neurological liabilities in conscious dogs in early drug development.

INTRODUCTION: Convulsions in toxicology studies can be the first indication of seizure liability. Drug levels during convulsions are not usually evaluated. This, and exposure variability after oral administration, complicates estimation of safety margins. The electroencephalogram (EEG) enables symptoms to be attributed to seizures and to collect samples during epileptiform activity without clinical convulsion. We evaluated an EEG-study design for optimized detection of neurological symptoms. Additionally, we assessed whether EEG- based anticonvulsive treatment is feasible, to prevent progression to convulsions and if dogs have higher sensitivity towards neurological symptoms than non-human-primates. METHODS: Three compounds that previously were tested in non-human-primates were selected to evaluate the dog EEG-study design. Two substances were administered in escalating intravenous doses; the third was given as single oral dose. Per compound, one male and one female telemetered dog were evaluated; males also had cerebrospinal-fluid-ports. Drug levels, video-EEG and clinical symptoms were evaluated and compared to previous studies. RESULTS: While similar neurological symptoms were induced, intravenous administration reduced experimental time compared to standard toxicology studies. EEG analysis could link animal behavior to seizures but did not allow convulsion prevention. This was due to artefacts and the short latency between onset of epileptiform EEG activity and clinical convulsions. Free plasma concentrations during convulsions were comparable between dogs and non-human-primates. DISCUSSION: The findings suggest that infusion studies provide a possibility to investigate neurological adverse effects in few animals in a short time period. For candidates with a high risk for seizures, such studies can guide dose selection for longer regulatory studies and improve safety margin definition.

Measuring drug absorption improves interpretation of behavioral responses in a larval zebrafish locomotor assay for predicting seizure liability.

INTRODUCTION: Unanticipated effects on the central nervous system are a concern during new drug development. A larval zebrafish locomotor assay can reveal seizure liability of experimental molecules before testing in mammals. Relative absorption of compounds by larvae is lacking in prior reports of such assays; having those data may be valuable for interpreting seizure liability assay performance. METHODS: Twenty-eight reference drugs were tested at multiple dose levels in fish water and analyzed by a blinded investigator. Responses of larval zebrafish were quantified during a 30min dosing period. Predictive metrics were calculated by comparing fish activity to mammalian seizure liability for each drug. Drug level analysis was performed to calculate concentrations in dose solutions and larvae. Fifteen drug candidates with neuronal targets, some having preclinical convulsion findings in mammals, were tested similarly. RESULTS: The assay has good predictive value of established mammalian responses for reference drugs. Analysis of drug absorption by larval fish revealed a positive correlation between hyperactive behavior and pro-convulsive drug absorption. False negative results were associated with significantly lower compound absorption compared to true negative, or true positive results. The predictive value for preclinical toxicology findings was inferior to that suggested by reference drugs. DISCUSSION: Disproportionately low exposures in larvae giving false negative results demonstrate that drug exposure analysis can help interpret results. Due to the rigorous testing commonly performed in preclinical toxicology, predicting convulsions in those studies may be more difficult than predicting effects from marketed drugs.